Alpha herpesvirus exocytosis from neuron cell bodies uses constitutive secretory mechanisms, and egress and spread from axons is independent of neuronal firing activity.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system, causing severe debilitating or deadly disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous activity, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. Using live-cell fluorescence microscopy, we show that Pseudorabies Virus (PRV) particles use the constitutive Rab6 post-Golgi secretory pathway to exit from the cell body of primary neurons, independent of local calcium signaling. Some PRV particles colocalize with Rab6 in the proximal axon, but we did not detect colocalization/co-transport in the distal axon. Thus, the specific secretory mechanisms used for viral egress from axons remains unclear. To address the role of neuronal activity more generally, we used a compartmentalized neuron culture system to measure the egress and spread of PRV from axons, and pharmacological and optogenetics approaches to modulate neuronal activity. Using tetrodotoxin to silence neuronal activity, we observed no inhibition, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread from axons. We conclude that PRV egress from neurons uses constitutive secretory mechanisms: generally, activity-independent mechanisms in axons, and specifically, the constitutive Rab6 post-Golgi secretory pathway in cell bodies.

Quantitative Characterization of Output from the Directionally Selective Visual Interneuron H1 in the Grey Flesh Fly Sarcophaga bullata.

H1, a very well-studied insect visual interneuron, has a panoramic receptive field and is directionally selective in responding to optic flow. The synaptic basis for the directional selectivity of the H1 neuron has been studied using both theoretical and cellular approaches. Extracellular single-unit recordings are readily obtained by beginning students using commercially available adults of the grey flesh fly Sarcophaga bullata. We describe an apparatus which allows students to present a series of moving visual stimuli to the eye of the restrained, minimally dissected adult Sarcophaga, while recording both the single unit responses of the H1 neuron and the position and velocity of the moving stimulus. Students obtain quantitative and reproducible responses of H1, probing the response properties of the neuron by modulating stimulus parameters such as: direction and speed of movement, visual contrast, spatial wavelength, or the extent of the visual field occupied. Students learn to perform quantitative analysis of their data and to generate graphical representations of their results characterizing the tuning and receptive field of this neuron. This exercise demonstrates the utility of single unit recording of an identified interneuron in an awake restrained insect and promotes interpretation of these results in terms of the visual stimuli normally encountered by freely flying flies in their natural environment.

Reproducible Quantitative Stimulation Allows New Analysis of Crayfish Muscle Receptor Organ Responses.

The crustacean muscle receptor organ (MRO) has provided a particularly accessible preparation for the study of sensory coding, which has been widely used in introductory laboratory courses incorporating extracellular recording from sensory nerves in living preparations. We describe three innovations to the standard laboratory exercise using the MRO: (1) a new form of suction electrode to facilitate extracellular recording; (2) a new, Arduino-driven actuator to allow reproducible and quantifiable mechanical stimulation of the MRO; and (3) a new approach to the crayfish abdomen preparation that allows linear extension of the MRO muscles. These novel approaches allow the collection of data sets comprised of sensory cell spike trains under software control as important mechanical stimulus parameters are varied systematically through software. This additional level of user control facilitates a more robust quantitative approach to the analysis of MRO sensory neuron spike trains, which is facilitated by training in data analysis using python.

Kinesin-3 mediates axonal sorting and directional transport of alphaherpesvirus particles in neurons.

During infection of the nervous system, alphaherpesviruses-including pseudorabies virus (PRV)-use retrograde axonal transport to travel toward the neuronal cell body and anterograde transport to traffic back to the cell periphery upon reactivation from latency. The PRV protein Us9 plays an essential but unknown role in anterograde viral spread. To determine Us9 function, we identified viral and host proteins that interact with Us9 and explored the role of KIF1A, a microtubule-dependent kinesin-3 motor involved in axonal sorting and transport. Viral particles are cotransported with KIF1A in axons of primary rat superior cervical ganglion neurons, and overexpression or disruption of KIF1A function, respectively, increases and reduces anterograde capsid transport. Us9 and KIF1A interact early during infection with the aid of additional viral protein(s) but exhibit diminished binding at later stages, when capsids typically stall in axons. Thus, alphaherpesviruses repurpose the axonal transport and sorting pathway to spread within their hosts.

Fine-mapping subtelomeric deletions and duplications by comparative genomic hybridization in 42 individuals.

Human subtelomere regions contain numerous gene-rich segments and are susceptible to germline rearrangements. The availability of diagnostic test kits to detect subtelomeric rearrangements has resulted in the diagnosis of numerous abnormalities with clinical implications including congenital heart abnormalities and mental retardation. Several of these have been described as clinically recognizable syndromes (e.g., deletion of 1p, 3p, 5q, 6p, 9q, and 22q). Given this, fine-mapping of subtelomeric breakpoints is of increasing importance to the assessment of genotype-phenotype correlations in these recognized syndromes as well as to the identification of additional syndromes. We developed a BAC and cosmid-based DNA array (TEL array) with high-resolution coverage of 10 Mb-sized subtelomeric regions, and used it to analyze 42 samples from unrelated patients with subtelomeric rearrangements whose breakpoints were previously either unmapped or mapped at a lower resolution than that achievable with the TEL array. Six apparently recurrent subtelomeric breakpoint loci were localized to genomic regions containing segmental duplication, copy number variation, and sequence gaps. Small (1 Mb or less) candidate gene regions for clinical phenotypes in separate patients were identified for 3p, 6q, 9q, and 10p deletions as well as for a 19q duplication. In addition to fine-mapping nearly all of the expected breakpoints, several previously unidentified rearrangements were detected.

Human subtelomeric duplicon structure and organization.

BACKGROUND: Human subtelomeric segmental duplications ('subtelomeric repeats') comprise about 25% of the most distal 500 kb and 80% of the most distal 100 kb in human DNA. A systematic analysis of the duplication substructure of human subtelomeric regions was done in order to develop a detailed understanding of subtelomeric sequence organization and a nucleotide sequence-level characterization of subtelomeric duplicon families. RESULTS: The extent of nucleotide sequence divergence within subtelomeric duplicon families varies considerably, as does the organization of duplicon blocks at subtelomere alleles. Subtelomeric internal (TTAGGG)n-like tracts occur at duplicon boundaries, suggesting their involvement in the generation of the complex sequence organization. Most duplicons have copies at both subtelomere and non-subtelomere locations, but a class of duplicon blocks is identified that are subtelomere-specific. In addition, a group of six subterminal duplicon families are identified that, together with six single-copy telomere-adjacent segments, include all of the (TTAGGG)n-adjacent sequence identified so far in the human genome. CONCLUSION: Identification of a class of duplicon blocks that is subtelomere-specific will facilitate high-resolution analysis of subtelomere repeat copy number variation as well as studies involving somatic subtelomere rearrangements. The significant levels of nucleotide sequence divergence within many duplicon families as well as the differential organization of duplicon blocks on subtelomere alleles may provide opportunities for allele-specific subtelomere marker development; this is especially true for subterminal regions, where divergence and organizational differences are the greatest. These subterminal sequence families comprise the immediate cis-elements for (TTAGGG)n tracts, and are prime candidates for subtelomeric sequences regulating telomere-specific (TTAGGG)n tract length in humans.

Mapping and initial analysis of human subtelomeric sequence assemblies.

Physical mapping data were combined with public draft and finished sequences to derive subtelomeric sequence assemblies for each of the 41 genetically distinct human telomere regions. Sequence gaps that remain on the reference telomeres are generally small,well-defined,and for the most part,restricted to regions directly adjacent to the terminal (TTAGGG)n tract. Of the 20.66 Mb of subtelomeric DNA analyzed, 3.01 Mb are subtelomeric repeat sequences (Srpt),and an additional 2.11 Mb are segmental duplications. The subtelomeric sequence assemblies are enriched >25-fold in short,internal (TTAGGG)n-like sequences relative to the rest of the genome; a total of 114 (TTAGGG)n-like islands were found,55 within Srpt regions,35 within one-copy regions,11 at one-copy/Srpt or Srpt/segmental duplication boundaries,and 13 at the telomeric ends of assemblies. Transcripts were annotated in each assembly,noting their mapping coordinates relative to their respective telomere and whether they originate in duplicated DNA or single-copy DNA. A total of 697 transcripts were found in 15.53 Mb of one-copy DNA,76 transcripts in 2.11 Mb of segmentally duplicated DNA,and 168 transcripts in 3.01 Mb of Srpt sequence. This overall transcript density is similar (within approximately 10%) to that found genome-wide. Zinc finger-containing genes and olfactory receptor genes are duplicated within and between multiple telomere regions.